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1.
Nat Commun ; 14(1): 4219, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37452020

RESUMO

Recent analyses of public microbial genomes have found over a million biosynthetic gene clusters, the natural products of the majority of which remain unknown. Additionally, GNPS harbors billions of mass spectra of natural products without known structures and biosynthetic genes. We bridge the gap between large-scale genome mining and mass spectral datasets for natural product discovery by developing HypoRiPPAtlas, an Atlas of hypothetical natural product structures, which is ready-to-use for in silico database search of tandem mass spectra. HypoRiPPAtlas is constructed by mining genomes using seq2ripp, a machine-learning tool for the prediction of ribosomally synthesized and post-translationally modified peptides (RiPPs). In HypoRiPPAtlas, we identify RiPPs in microbes and plants. HypoRiPPAtlas could be extended to other natural product classes in the future by implementing corresponding biosynthetic logic. This study paves the way for large-scale explorations of biosynthetic pathways and chemical structures of microbial and plant RiPP classes.


Assuntos
Produtos Biológicos , Ribossomos , Ribossomos/metabolismo , Produtos Biológicos/química , Peptídeos/química , Bases de Dados Factuais , Espectrometria de Massas em Tandem , Processamento de Proteína Pós-Traducional
2.
Biochemistry ; 62(7): 1233-1248, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-36917754

RESUMO

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward ß-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here, we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and GoA with and without the brain-penetrant small-molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are Gα subunit selective and positive allosteric modulation and agonism for ß-arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1.


Assuntos
Neurotensina , Receptores de Neurotensina , Receptores de Neurotensina/química , Receptores de Neurotensina/metabolismo , Neurotensina/metabolismo , Transdução de Sinais , Peptídeos/metabolismo , beta-Arrestinas/metabolismo
3.
Nature ; 613(7945): 767-774, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36450356

RESUMO

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.


Assuntos
Desenho de Fármacos , Fentanila , Morfinanos , Receptores Opioides mu , Animais , Camundongos , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Arrestinas/metabolismo , Microscopia Crioeletrônica , Fentanila/análogos & derivados , Fentanila/química , Fentanila/metabolismo , Ligantes , Morfinanos/química , Morfinanos/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Receptores Opioides mu/ultraestrutura , Sítios de Ligação , Nociceptividade
4.
Nature ; 610(7932): 582-591, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36171289

RESUMO

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally1-4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT2A receptor (5-HT2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT2A or the 5-HT2B receptors. Structure-based optimization led to the 5-HT2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.


Assuntos
Antidepressivos , Pirrolidinas , Receptor 5-HT2A de Serotonina , Animais , Camundongos , Antidepressivos/farmacologia , Microscopia Crioeletrônica , Fluoxetina/administração & dosagem , Fluoxetina/farmacologia , Alucinógenos/administração & dosagem , Alucinógenos/farmacologia , Ligantes , Pirrolidinas/administração & dosagem , Pirrolidinas/farmacologia , Receptor 5-HT2A de Serotonina/metabolismo , Bibliotecas de Moléculas Pequenas
5.
ACS Chem Neurosci ; 13(16): 2386-2398, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35894503

RESUMO

Serotonin (5-hydroxytryptamine; 5-HT) 2A receptor (5-HT2AR) signaling is essential for the actions of classical psychedelic drugs. In this study, we examined whether sequence variations in the 5-HT2AR gene affect the signaling of four commonly used psychedelic drugs. We examined the in vitro pharmacology of seven non-synonymous single-nucleotide polymorphisms (SNPs), which give rise to Ser12Asn, Thr25Asn, Asp48Asn, Ile197Val4.47, Ala230Thr, Ala447Val, and His452Tyr variant 5-HT2A serotonin receptors. We found that these non-synonymous SNPs exert statistically significant, although modest, effects on the efficacy and potency of four therapeutically relevant psychedelics. Significantly, the in vitro pharmacological effects of the SNP drug actions at 5-HT2AR are drug specific.


Assuntos
Alucinógenos , Alucinógenos/farmacologia , Polimorfismo de Nucleotídeo Único/genética , Receptor 5-HT2A de Serotonina/genética , Serotonina , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais
6.
J Neurochem ; 162(1): 24-38, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34797943

RESUMO

A confluence of factors has renewed interest in the scientific understanding and translational potential of psychedelic drugs such as lysergic acid diethylamide (LSD), mescaline, and psilocybin: the desire for additional approaches to mental health care, incremental progress in basic and clinical research, and the reconsideration and relaxation of existing drug policies. With the United States Food and Drug Administration's designation of psilocybin as a "Breakthrough Therapy" for treatment-resistant depression, a new path has been forged for the conveyance of psychedelics to the clinic. Essential to the further development of such applications, however, is a clearer understanding of how these drugs exert their effects at the molecular level. Here we review the current knowledge regarding the molecular details of psychedelic drug actions and suggest that these discoveries can facilitate new insights into their hallucinogenic and therapeutic mechanisms.


Assuntos
Alucinógenos , Alucinógenos/farmacologia , Alucinógenos/uso terapêutico , Dietilamida do Ácido Lisérgico/farmacologia , Dietilamida do Ácido Lisérgico/uso terapêutico , Psilocibina/farmacologia , Psilocibina/uso terapêutico , Estados Unidos
7.
Nature ; 600(7887): 170-175, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34789874

RESUMO

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.


Assuntos
Microscopia Crioeletrônica , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Prurido/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/química , Agonismo Inverso de Drogas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/ultraestrutura
8.
J Med Chem ; 64(22): 16553-16572, 2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34783240

RESUMO

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.


Assuntos
Alcaloides de Triptamina e Secologanina/farmacologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Receptores Opioides mu
9.
Elife ; 102021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33555255

RESUMO

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.


Assuntos
Morfinanos/química , Receptores Opioides kappa/química , Receptores Opioides mu/química , Motivos de Aminoácidos , Analgésicos/química , Analgésicos/metabolismo , Animais , Sítios de Ligação , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Relação Estrutura-Atividade
10.
Nat Neurosci ; 23(9): 1157-1167, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32632286

RESUMO

The chemogenetic technology designer receptors exclusively activated by designer drugs (DREADDs) afford remotely reversible control of cellular signaling, neuronal activity and behavior. Although the combination of muscarinic-based DREADDs with clozapine-N-oxide (CNO) has been widely used, sluggish kinetics, metabolic liabilities and potential off-target effects of CNO represent areas for improvement. Here, we provide a new high-affinity and selective agonist deschloroclozapine (DCZ) for muscarinic-based DREADDs. Positron emission tomography revealed that DCZ selectively bound to and occupied DREADDs in both mice and monkeys. Systemic delivery of low doses of DCZ (1 or 3 µg per kg) enhanced neuronal activity via hM3Dq within minutes in mice and monkeys. Intramuscular injections of DCZ (100 µg per kg) reversibly induced spatial working memory deficits in monkeys expressing hM4Di in the prefrontal cortex. DCZ represents a potent, selective, metabolically stable and fast-acting DREADD agonist with utility in both mice and nonhuman primates for a variety of applications.


Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Clozapina/análogos & derivados , Drogas Desenhadas/farmacologia , Neurônios/efeitos dos fármacos , Animais , Clozapina/farmacologia , Técnicas Genéticas , Humanos , Macaca fuscata , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Receptor Muscarínico M3/metabolismo , Receptor Muscarínico M4/metabolismo
11.
Nat Chem Biol ; 16(8): 841-849, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32367019

RESUMO

G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαßγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαßγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.


Assuntos
Técnicas Biossensoriais/instrumentação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Técnicas Biossensoriais/métodos , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Engenharia de Proteínas/métodos , Transdução de Sinais
12.
J Med Chem ; 62(16): 7557-7574, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31298539

RESUMO

G protein-coupled receptor 68 (GPR68) is an understudied orphan G protein-coupled receptor (GPCR). It is expressed most abundantly in the brain, potentially playing important roles in learning and memory. Pharmacological studies with GPR68 have been hindered by lack of chemical tools that can selectively modulate its activity. We previously reported the first small-molecule positive allosteric modulator (PAM), ogerin (1), and showed that 1 can potentiate proton activity at the GPR68-Gs pathway. Here, we report the first comprehensive structure-activity relationship (SAR) study on the scaffold of 1. Our lead compound resulted from this study, MS48107 (71), displayed 33-fold increased allosteric activity compared to 1. Compound 71 demonstrated high selectivity over closely related proton GPCRs and 48 common drug targets, and was bioavailable and brain-penetrant in mice. Thus, our SAR study has resulted in an improved GPR68 PAM for investigating the physiological and pathophysiological roles of GPR68 in vitro and in vivo.


Assuntos
Regulação Alostérica , Álcoois Benzílicos/farmacologia , Desenho de Fármacos , Prótons , Receptores Acoplados a Proteínas G/metabolismo , Triazinas/farmacologia , Animais , Álcoois Benzílicos/síntese química , Álcoois Benzílicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Cálcio/metabolismo , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Ensaio Radioligante , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/farmacocinética
13.
14.
Nature ; 569(7755): 289-292, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31019305

RESUMO

The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.


Assuntos
Elétrons , Lasers , Modelos Moleculares , Receptor MT2 de Melatonina/química , Receptor MT2 de Melatonina/metabolismo , Cristalização , Diabetes Mellitus Tipo 2/genética , Humanos , Indenos/química , Indenos/metabolismo , Ligantes , Melatonina/análogos & derivados , Melatonina/química , Melatonina/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Relação Estrutura-Atividade , Especificidade por Substrato
15.
Nature ; 569(7755): 284-288, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31019306

RESUMO

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.


Assuntos
Elétrons , Lasers , Modelos Moleculares , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Sequência de Aminoácidos , Antidepressivos/química , Antidepressivos/metabolismo , Cristalização , Humanos , Indenos/química , Indenos/metabolismo , Ligantes , Melatonina/análogos & derivados , Melatonina/química , Simulação de Acoplamento Molecular , Mutação , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/genética , Receptor 5-HT2C de Serotonina/química , Relação Estrutura-Atividade , Especificidade por Substrato
16.
Methods Enzymol ; 604: 207-236, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29779653

RESUMO

ß-Branching is an expansion upon canonical polyketide synthase extension that allows for the installation of diverse chemical moieties in several natural products. Several of these moieties are unique among natural products, including the two vinyl methylesters found in the core structure of bryostatins. This family of molecules is derived from an obligate bacterial symbiont of a sessile marine bryozoan, Bugula neritina. Within this family, bryostatin 1 has been investigated as an anticancer, neuroprotective, and immunomodulatory compound. We have turned to the biosynthetic gene cluster within the bacterial symbiont to investigate the biosynthesis of bryostatins. Recent sequencing efforts resulted in the annotation of two missing genes: bryT and bryU. Using novel chemoenzymatic techniques, we have validated these as the missing enoyl-CoA hydratase and donor acyl carrier protein, essential components of the ß-branching cassette of the bryostatin pathway. Together, this cassette installs the vinyl methylester moieties essential to the activity of bryostatins.


Assuntos
Bioquímica/métodos , Briostatinas/metabolismo , Enzimas/metabolismo , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Animais , Briostatinas/biossíntese , Briozoários/genética , Briozoários/metabolismo , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , Enzimas/genética , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas , Metilação , Família Multigênica , Policetídeos/metabolismo
17.
J Am Chem Soc ; 139(23): 7913-7920, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28525276

RESUMO

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.


Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Tilosina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Macrolídeos/química , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Conformação Molecular , Policetídeo Sintases/química , Policetídeos/química , Policetídeos/farmacologia , Relação Estrutura-Atividade , Tilosina/biossíntese , Tilosina/química , Tilosina/farmacologia
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